Photodynamic therapy (PDT) involves the administration of a photosensitising agent for localisation in target diseased tissue followed by irradiation of the target tissue containing the compound with light of a specific and appropriate wavelength. The resulting photoactivated compound, in the presence of oxygen, leads to necrosis of the tissue.
The success of this modality is dependent on administration of a compound that is selectively retained in tumour tissue as compared to normal tissue. Thus, on irradiation of the tumour with light of the photoactivating wavelength, the amount of damage caused by necrosis is proportionately higher than that in normal tissue. However, some normal tissue damage typically occurs and one specific side effect seen with the use of many photosensitisers is redness and swelling of the skin on subsequent exposure to normal lighting levels and particularly sunlight. Such side effects are minimised by keeping the patients in subdued light for a prolonged period after treatment, consequently restricting their quality of life. A more efficient delivery of the photosensitiser into tumour tissue, thus providing a much higher tumour to normal tissue ratio of drug concentration could dramatically reduce the potential for skin side effects with this treatment.
A group of photosensitising agents have previously been the subject of patents EP 0 337 601 and U.S. Pat. No. 4,992,257. These compounds are dihydroporphyrins (chlorins) (1) and the corresponding tetrahydroporphyrins (bacteriochlorins) (2) and (3) of the formulae:

wherein each n=1 to 3 and each substituent, R, the same or different, is a hydroxyl (—OH) group, each itself free or substituted with an alkyl or acyl group. Salts, internal salts, metal complexes or hydrates or other solvates of the compounds are also covered.
The above formulae, it will be appreciated, represent particular tautomers among various possibilities including chlorins as shown below (represented without meso Phenyl groups):

The invention covers all tautomers of the above compounds and is not limited to those shown in diagrams.
Published Proposals
Modification of compounds by PEGylation, that is the direct or indirect attachment of polyethylene glycol chains (PEG), and in principle other poly(alkylene oxide) chains, is known to introduce useful properties. PEG is non-toxic, imparts good water solubility to drug molecules and alters the biodistribution, which can result in a favourable pharmacokinetic profile. The general topic of polyether substituted anti-tumour agents is described in DKFZ's specification PCT EP 91/00992 (WO 91/18630). No particular attention is given to the selection of the linkage between the polyether chain and the anti-tumour agent, the only example disclosed being a triazine introduced by initial activation of the polyether with cyanuric chloride. More recently, DKFZ have described a method for the production of chlorins and bacteriochlorins containing a polyether (WO 98/01156). The method involves initial attachment of the polyether to the porphyrin with subsequent reduction to the corresponding chlorins and bacteriochlorins. Again, no particular attention is given to the nature of the linkage between the polyether and the anti-tumour agent, the only example disclosed being an amide link.
PEGylation of compounds (1), (2) and (3) via triazine, ether and ester linkages has been previously reported by us in PCT GB 95/00998 (WO 95/29915). However, lability of the ester linkages and significant difficulties in the scale up of the triazine and ether linked moieties severely limits the practical utility of these compounds.
Enzon have also reported polyether compositions containing isocyanate and/or isothiocyanate groups for covalent attachment to bioeffecting substances such as peptides or chemotherapeutics (WO 94/04193). However, in relation to isocyanates and isothiocyanates, coverage is directed to compounds in which bioeffecting substances are attached to both ends of the polyether chain.
Outside the PDT field, hexane-1,6-diisocyanate has been used to link PEG to atropine (Zalipsky et al, Eur. Polym. J. 1983, 19(12), 1177-1183) and to 5-fluorouracil (Ouchi et al, Drug Design and Discovery 1992, 9, 93-105). Bayer (U.S. Pat. No. 4,684,728) have reported a process for improving the solubility in water of a sparingly soluble biologically active compound by reaction to form a derivative carrying the active moiety, a linking group such as an optionally substituted diisocyanate group and a polyether chain. No mention is made of any benefit to the therapeutic profile of such compounds other than the ease of formulation and administration of a water soluble compound.
Discussion of Present Work
Advances in photodynamic therapy for clinical disease treatment, particularly cancer, depend on developing improved photosensitisers. The desired characteristics of an ideal photosensitiser include selective diseased tissue localisation, activation at long wavelengths so that maximum depth of tissue penetration is shown, and high efficiency as sensitisers. From a formulation and administration point of view, water solubility is also a beneficial attribute.
Photodynamic therapy is a dual therapy, which consists of the combined action of photosensitiser and light. In clinical practice the drug is first administered, and then activated by light some time later. The time period between administering the drug and applying the light is called the drug-light interval. It is desirable to apply light at a time when the photosensitiser has accumulated maximally in the target tissue and has been eliminated from the normal surrounding tissue. The principal factor determining the drug-light interval is the drug pharmacokinetic profile which itself varies between every tissue. The drug-light interval has to be suitable for clinical practice. From a clinical standpoint, pharmacokinetics which give a maximum drug concentration in a tumour as soon as possible, for example from a few hours to at most 3 days, together with rapid elimination from the body thereafter, would be ideal. This would allow flexibility in scheduling treatment.
In European Patent Specification 0 337 601 (U.S. Pat. No. 4,992,257), the applicants disclose compounds with many of the desired characteristics, particularly an extremely high photo efficiency, that is to say the ability to generate free radical species such as singlet oxygen through the absorption of light. The long absorption wavelengths of the molecules, e.g. at 652 nm and 734 nm, penetrates tissue efficiently and thus the sensitisers can be used to treat deep tumours.
However, the disclosed compounds do not fulfil all the requirements equally well. A residual disadvantage is the degree of normal tissue photosensitivity, particularly of the skin, that occurs following administration of the sensitiser. This arises from unwanted deposition of the sensitiser in the skin and other normal tissue and is a consequence of imperfect tumour targeting by the drug. The skin photosensitivity can last up to 4 weeks depending on the drug dose administered. At the usual clinical dose of 0.15 mg kg−1 skin sensitivity of, for example, m-THPC (a tetraphenyl chlorin derivative in which each phenyl group carries a m-hydroxy group) lasts for 2-3 weeks. This limits the patient's freedom and is an undesirable restriction.
We have sought ways of overcoming normal tissue photosensitivity, particularly of the skin, by converting m-THPC to a polyethylene glycol derivative. This ‘PEGylation’ profoundly alters the bodily distribution in a favourable way by increasing tumour targeting, and at the same time reducing uptake to the skin. The distribution of the compound is altered by PEGylation, due to hydrogen bonding of water to oxygen on the polyethylene glycol chains when the compound is injected into the blood. A ‘water envelope’ forms around the photosensitiser and prevents the compound sticking to the endothelium of blood vessel walls and in turn passing into the surrounding tissue including the skin. This favours uptake to the tumour through the enhanced permeability and retention (EPR) effect. Tumours have a disturbed vasculature and lymphatic drainage, leading to increased accumulation of substances such as drugs in the tumour compared to normal tissue (R. Duncan and F. Spreafico, Clin. Pharmacokinet. 1994, 27, 290-306). This effect can be enhanced with higher molecular weight compounds. The net effect of
PEGylation is that the compound is favourably redirected from the skin and other normal tissues towards the tumour, thus reducing the degree of skin sensitivity.
It has been confirmed experimentally, for example, that PEGylation can produce a favourable tissue re-distribution. This was shown in a mouse experimental model in which an outstanding difference between muscle and tumour photosensitivity during PDT was found for the triazine-linked derivative (Grahn et al., Proc. SPE 1997, 3191, 180-6). Three days after the PEGylated m-THPC was administered it was found that the muscle was no longer photosensitive, while the tumour retained its maximum sensitivity to light for at least 15 days after drug administration. This presented ample time for tumour-selective treatment, but did indicate the less desirable characteristic of tumour persistence with this derivative.
Other work previously performed with triazine-linked PEG derivatives [applicants PCT Patent specification WO 95/29915, (PCT/GB95/00998)] confirmed that the pharmacokinetics with the triazine linkage were rather too prolonged for routine clinical use. In particular, excretion from the liver was very slow indeed, which is undesirable pharmaceutically.
An alternative linker to the triazine molecule was sought, including a glycidic ether with amino PEG and also an hexylbiscarbamate linker. The PEGylated derivatives of m-THPC with triazine and carbamate links have very different and unexpected pharmacokinetics to each other and m-THPC. The triazine-linked compound (SPC 0038B) is excreted from the liver more slowly than m-THPC, while the carbamate derivative (SPC 0172) is excreted in a comparable period to m-THPC. The solubilities of the compounds, and hence ease of pharmaceutical preparation, were enhanced to levels of up to 52 mg/mL in water, compared to m-THPC, which is insoluble in aqueous solvents.
The linker group should provide a stable point of PEG attachment, permitting reasonable in vivo circulation and should be available via a practical and robust synthesis. It should not, however, affect the PDT efficiency of the drug molecule. The method of Zalipsky was modified and utilised for a two step synthesis from the chlorin and bacteriochlorin molecules to their PEGylated derivatives. Analysis of these compounds using gel permeation chromatography (GPC) allowed separation of lesser PEGylated forms, but high performance liquid chromatography (HPLC) proved superior with separation between the peaks of 0.8 min. Reaction products were also analysed by UV/Visible spectroscopy, which gave a quantitative measurement of molecular weight (mw) using the formula:Apparent mw=mw (chlorin)×A(1%, 1 cm) (chlorin)/A(1%, 1 cm) (PEG chlorin).
The applicants work has thus built on previous work in trying to develop an ideal sensitiser. Unpredictably, the carbamate-linked polyethylene glycol derivatives have an excellent and preferred pharmacokinetic profile from a clinical point of view and exhibit less potential to cause cutaneous photosensitivity. Furthermore, studies in Balb/c mice bearing colo26, a murine colorectal cancer, show that the photodynamic effect of the carbamate-linked derivative in tumour is maximum at 2 days and that it has the same PDT activity as m-THPC itself This is considerably more active than the triazine-linked compound. Thus overall, the PEGylated carbamate-linked compound appears to add new features, which enhance the desired characteristics of the sensitiser.
The Invention
The present invention summarised below and set out in the claims thus concerns the derivatisation or partial derivatisation of the phenolic groups of compounds of formulae (1), (2) and (3) with poly(alkylene oxides) using a carbamate or thiocarbamate link:

(X═O, S) formed by addition reaction of the compounds with an isocyanate (—N═C═O) or isothiocyanate (—N═C═S) group of a diisocyanate, diisothiocyanate or an isocyanate-isothiocyanate, the poly(alkylene oxide) chain being attached directly or indirectly by addition at the other group and its terminal hydroxyl group being etherified or esterified with for example a C1-12 alkyl or acyl group of which methyl is the most preferred.
The reactions, which may be carried out in any convenient order, result in compounds of formulae:

and imino-tautomers thereof wherein n=1 to 3 and R′ may be the same or different, is a hydroxyl (—OH) group, each itself free or substituted with an alkyl or acyl group, but in at least one, preferably more than one instance is as follows:

where:                (i) each X, the same or different, is O, S;        (ii) Y is O (carbamate or thiocarbamate link);        (iii) A is a hydrocarbon group containing 2 to 40 carbon atoms, preferably 4 to 20 carbon atoms and very preferably 6 carbon atoms. This group may be branched or unbranched, cyclic or acyclic, saturated or unsaturated, aliphatic or aromatic;        (iv) B is an optional group ((CH2)p—O)q where p=1 to 4; q=0,1;        (v) D is a poly(alkylene oxide), preferably polyethylene glycol, with an average molecular weight of at least 200 and not more than 40,000, preferably 750 to 20,000 and very preferably 2,000 to 5,000 Da;        (vi) E is an alkyl or acyl group containing 1 to 12 carbon atoms, preferably a methyl group.        
In any of the above compounds derivatives such as salts with mineral acids (e.g. hydrochlorides, sulphates), internal salts, metal complexes (e.g. with Zn, Ga), or hydrates and other solvates may be formed.
Suitable diisocyanates include butane-1,4-diisocyanate, hexane-1,6-diisocyanate, octane-1,8-diisocyanate, dodecane-1,1 2-iisocyanate, 2-methylpentane-1,5-diisocyanate, toluene-2,4-diisocyanate, toluene-2,6-diisocyanate, cyclohexane-trans-1,4diisocyanate, dicyclohexylmethane-4,4′-diisocyanate, diphenylmethane-3,4′-diisocyanate, xylene diisocyanate and 2,4,4-trimethylhexylmethylene diisocyanate. Corresponding diisothiocyanates and isocyanate-isothiocyanates are also appropriate. The most preferred linker is hexane-1,6-diisocyanate.
Chemistry
Compounds of types (12), (13) and (14) may be prepared in a two step process.    (i) activation of poly(alkylene oxide) by reaction with a diisocyanate, diisothiocyanate or an isocyanate-isothiocyanate (e.g. hexane-1,6-diisocyanate) in a suitable inert, anhydrous solvent (e.g. toluene) with or without a catalyst (e.g. dibutyl tin dilaurate), with or without a tertiary organic base (e.g. triethylamine) at a temperature between 0 and 110° C.    (ii) coupling of the activated poly(alkylene oxide) to the reduced porphyrin in a suitable inert solvent (e.g. toluene) with or without a catalyst (e.g. dibutyl tin dilaurate), with or without a tertiary organic base (e.g. triethylamine) at a temperature between 0 and 110° C.
Synthesis of compounds may also be achieved by reversing the order of the steps, namely activation of the reduced porphyrin by reaction with the diisocyanate, diisothiocyanate or isocyanate-isothiocyanate followed by coupling with the poly(alkylene oxide). However, the former approach is preferred.
Routes of Administrations
By parenteral or any other suitable route in per se known manner.
Pharmaceutical Presentations
Any suitable presentation as known in the field, including, but not limited to:                i) injectable solution        ii) freeze dried powder for reconstitution and injection        iii) infusion solution for addition to saline or other vehicle        iv) tablet or capsule for oral administration.        